Antibody-blocking of a tick transporter impairs Anaplasma phagocytophilum colonization in Haemaphysalis longicornis ticks

The invasive Asian longhorned tick Haemaphysalis longicornis that vectors and transmits several animal pathogens is significantly expanding in the United States. Recent studies report that these ticks also harbor human pathogens including Borrelia burgdorferi sensu lato, Babesia microti, and Anaplasma phagocytophilum. Therefore, studies that address the interactions of these ticks with human pathogens are important. In this study, we report the characterization of H. longicornis organic anion-transporting polypeptides (OATPs) in interactions of these ticks with A. phagocytophilum. Using OATP-signature sequence, we identified six OATPs in the H. longicornis genome. Bioinformatic analysis revealed that H. longicornis OATPs are closer to other tick orthologs rather than to mammalian counterparts. Quantitative real-time PCR analysis revealed that OATPs are highly expressed in immature stages when compared to mature stages of these ticks. In addition, we noted that the presence of A. phagocytophilum upregulates a specific OATP in these ticks. We also noted that exogenous treatment of H. longicornis with xanthurenic acid, a tryptophan metabolite, influenced OATP expression in these ticks. Immunoblotting analysis revealed that antibody generated against Ixodes scapularis OATP cross-reacted with H. longicornis OATP. Furthermore, treatment of H. longicornis with OATP antibody impaired colonization of A. phagocytophilum in these ticks. These results not only provide evidence that the OATP-tryptophan pathway is important for A. phagocytophilum survival in H. longicornis ticks but also indicate OATP as a promising candidate for the development of a universal anti-tick vaccine to target this bacterium and perhaps other rickettsial pathogens of medical importance.

OATPs have a signature motif (WxGxWWxG) in the amino acid sequence 41 .We first screened the genome of H. longicornis and identified six OATPs (Supplementary Table 1).To analyze whether H. longicornis ticks express these OATPs, RNA from unfed uninfected adult ticks was used as a template for reverse transcription polymerase chain reaction (RT-PCR).Using oligonucleotides mentioned in Supplementary Table 2, we amplified all six OATP transcripts (Fig. 1A).The RT-PCR products were excised and sequenced to confirm the nucleotide sequence of all six OATPs.Amino acid percent identity analysis of H. longicornis OATP sequence with other ortholog proteins from Dermacentor andersoni (Da), Hyalomma asiaticum (Ha), Homo sapiens (Hs), I. scapularis (Is), Mus musculus (Mm) and Rhipicephalus sanguineus (Rs) was determined by CLUSTALW alignment (Fig. 1B).The GenBank accession numbers for these proteins are shown in Supplementary Table 3.The H. longicornis OATPs (KAH9381028.1,KAH9381027.1, KAH9381876.1, KAH9365504.1, KAH9380884.1, KAH9381025.1) share 62-88% identity with OATPs from other ticks analyzed in this study but 24-42% identity with OATPs from human and mice (Fig. 1B, Supplementary Figs. 1 and 2).Furthermore, phylogenetic analyses revealed that H. longicornis OATP amino acid sequences fall in the same clade or clade close to OATPs from other ticks (Supplementary Figs. 3 and 4).Both human and mouse OATPs form a different clade (Supplementary Figs. 3 and 4).These results reveal that H. longicornis OATPs are similar to OATPs from other ticks.

Comparison of OATP-signature sequence and prediction of posttranslational modifications on H. longicornis OATPs
The alignment of the amino acid sequences of H. longicornis OATPs with the OATP signature sequence WxGxWWxG 41 using ClustalW program revealed a conserved OATP signature motif in all six H. longicornis OATPs (Fig. 2A).Bioinformatic analysis of post translational modification sites in H. longicornis OATPs revealed that KAH9381027.1 had higher number of N-myristoylation sites (20 sites) and cAMP-and cGMP-dependent phospho sites (five sites) compared to other OATPs (Fig. 2B).KAH9381876.1 OATP had higher number of casein kinase II phospho sites (15 sites) and N-glycosylation (eight sites).KAH9381025.1 had the higher number of protein kinase C phospho sites (14 sites) compared to other OATPs.All but XP_040071371 of I. scapularis OATP orthologs had at least one of each of these posttranslational modification sites (Supplementary Table 4).Collectively, these analyses show that like I. scapularis, H. longicornis OATPs also have several posttranslational modification sites in their amino acid sequences.

Generation of A. phagocytophilum-infected H. longicornis ticks in vitro
Pathogens like Ehrlichia chaffeensis, A. bovis 11 , Lyme spirochetes and spotted fever group rickettsiae 12 have been detected in H. longicornis ticks.Recently, human pathogenic A. phagocytophilum variant was detected in field collected H. longicornis ticks in USA 8,9 .We reasoned whether expression of OATPs in H. longicornis is altered upon A. phagocytophilum infection.Anaplasma phagocytophilum-infected H. longicornis ticks were generated  3. in vitro as shown in the schematic diagram (Fig. 4A).Briefly, dense core (DC) form of A. phagocytophilum was isolated from infected HL-60 cultures as described 42 .Uninfected unfed H. longicornis nymphal ticks were bathed in A. phagocytophilum DC culture for 40 min (Fig. 4A).Ticks were washed and incubated for 3, 5, 7 and 10 days and processed for DNA extraction.qPCR was performed with the DNA isolated from these bathed and washed ticks to detect A. phagocytophilum loads.The qPCR analysis revealed no significant (P > 0.05) differences in the bacterial loads between day 3 and day 5 post-infected ticks (Fig. 4B).However, significantly (P < 0.05) higher bacterial loads were evident in both day 7 and day 10 post-infected ticks in comparison to day 3 and day 5 postinfected ticks (Fig. 4B).No significant (P > 0.05) differences in the bacterial loads were evident between day 7 and day 10 post-infected (p.i.) ticks (Fig. 4B).The results not only indicate day 7 as a peak of infection time point in the in vitro A. phagocytophilum infection of H. longicornis ticks but also suggests that A. phagocytophilum multiply in H. longicornis ticks.
We then used I. scapularis ticks which is a known vector for this pathogen as a control group and performed in vitro infection like the one performed with H. longicornis ticks.The in vitro infected I. scapularis was incubated for 7 days post infection.qPCR analysis followed by agarose gel electrophoresis confirmed that like H. longicornis, I. scapularis ticks could also be infected with A. phagocytophilum by bathing these ticks in isolated DC cultures (Supplementary Fig. 5A, B).However, qPCR analysis revealed no significant difference in the A. phagocytophilum loads between I. scapularis and H. longicornis ticks at day 7 p.i. (Supplementary Fig. 5B).

Xanthurenic acid (XA), a tryptophan metabolite, induces some of the OATPs expression in A. phagocytophilum-infected H. longicornis unfed nymphal ticks
In our previous study, we noted higher levels of XA in A. phagocytophilum-infected I. scapularis ticks 37 .Therefore, we studied whether XA has any impact on bacterial burden and OATP expression in A. phagocytophilum-infected H. longicornis ticks.First, we generated A. phagocytophilum-infected H. longicornis ticks (day 7 post-infected) as described in Fig. 4A.These A. phagocytophilum-infected H. longicornis nymphs were bathed in 100 µM XA or mock solutions as depicted in the schematic diagram (Fig. 5A).After 24 h, post-bathed ticks were washed and processed for DNA or RNA extractions.qPCR with DNA samples revealed significantly (P < 0.05) increased bacterial burden in XA-treated A. phagocytophilum-infected H. longicornis ticks compared to the levels noted in mock-treated control ticks (Fig. 5B).qRT-PCR analysis with RNA samples revealed no significant differences in the expression levels of kah9381027.1 (Fig. 5D) between mock-and XA-treated A. phagocytophilum-infected ticks.However, expression of kah9381876.1 (Fig. 5E), kah9365504.1 (Fig. 5F) and kah9380884.1 (Fig. 5G) were significantly (P < 0.05) upregulated in XA-treated A. phagocytophilum-infected H. longicornis ticks in comparison to the mock-treated controls.Conversely, expression of kah9381028.1 (Fig. 5C) and kah9381025.1 (Fig. 5H) were significantly (P < 0.05) down-regulated in XA-treated A. phagocytophilum-infected H. longicornis ticks in comparison to the respective mock-treated controls.These results show that XA modulates expression of OATPs in A. phagocytophilum-infected H. longicornis ticks.
In this study, we noted that kah9381876.1, was significantly upregulated upon A. phagocytophilum infection in H. longicornis ticks (Fig. 4E).The epitope for EL-6 antibody binding in I. scapularis IsOATP4056 36 is highly conserved in H. longicornis KAH9381876.1.Therefore, we used an anti-EL-6 antibody and performed immunoblotting and antibody-blocking experiments.Immunoblotting analyses with EL-6 antibody and 12% (Fig. 6B) Closed circles, squares and triangles represent larvae, nymphs and adults, respectively.For larval samples, each data point represents transcript levels noted in five pooled larvae.Horizontal lines in the graphs represent the mean value of the data points.The mRNA levels of these genes were normalized to tick beta-actin mRNA levels.Statistical significance was calculated using ANOVA analysis.
or 8% (Supplementary Fig. 6) SDS-PAGE performed in non-reducing (no boiling) and reducing conditions revealed an intense band above 250 kDa in H. longicornis (Hl) whole tick lysates (Fig. 6B).As expected, and based on our previous observation 36 , we noted intense band above 250 kDa in I. scapularis (Is) whole tick lysate (Fig. 6B and Supplementary Fig. 6).These results show that anti-EL-6 antibody also recognizes H. longicornis OATP protein (KAH9381876.1).We then explored whether treatment with anti-EL6 antibody has any effect on A. phagocytophilum burden in H. longicornis ticks.We noticed significant reduction in the bacterial burden in A. phagocytophilum-infected H. longicornis ticks treated with EL-6 antibody when compared to control IgGtreated groups (Fig. 6C).In summary, these results indicate that treatment with anti-EL-6 antibody impairs A. phagocytophilum colonization in H. longicornis ticks.

Discussion
Organic anion transporting polypeptides (OATPs) are a family of highly conserved transporters in arthropods like ticks, lice, and mosquitoes 41 .Ixodes scapularis tick express nine OATPs 41,43 .Our previous studies indicated that OATPs are critical for A. phagocytophilum survival and transmission from these ticks to the vertebrate host 34,36,37,39,40 .Ixodes scapularis and I. pacificus are the common vectors for A. phagocytophilum [17][18][19][20] .However, recent studies have indicated the presence of A. phagocytophilum in H. longicornis ticks collected from the field 8,9 .In this study, we noted that H. longicornis OATPs are also important for A. phagocytophilum survival in these ticks.The role of H. longicornis molecules in the interactions with A. phagocytophilum is not known.As OATPs are highly conserved in different ticks and are critical for A. phagocytophilum survival in I. scapularis ticks, we reasoned whether these important molecules have any role in the interactions of H. longicornis ticks with this bacterium.Screening of H. longicornis genome with OATP signature sequence (WXGXWWXG) resulted in the identification of six OATPs.Along with the conserved tryptophan and glycine residues, we noted that all six H. longicornis OATPs have conserved alanine residues in the OATP signature sequence.The significance of this conserved alanine residue in the OATP signature sequence is currently not understood.In addition to the six OATPs analyzed in this study, we also noted that a hypothetical protein (GenBank Acc.No. KAH9381873.1) was annotated as OATP.However, OATP signature sequence was absent in this sequence.Therefore, we excluded KAH9381873.1 from the current analysis.High degree of percent identity of H. longicornis OATPs with other tick OATPs, including I. scapularis, suggests a conserved functional role for these important molecules in various ticks.
We noted variable levels of OATP transcripts at different developmental stages of H. longicornis ticks.The observation of significantly lower trend of all analyzed H. longicornis OATP transcripts in adult ticks compared to the levels noted in larvae or nymphal stages of these ticks suggest that these molecules are important in the immature stages of ticks.Larval and nymphal H. longicornis ticks acquire pathogens by feeding on infected animals.In addition, OATPs are important for blood feeding 36,44 .Therefore, it could be reasoned that increased levels of H. longicornis OATP expression in larval or nymphal ticks could facilitate blood feeding and/or acquisition and transmission of pathogens from and to the vertebrate host, respectively.
Haemaphysalis longicornis OATPs contain several posttranslational sites in their amino acid sequences including glycosylation sites.In our previous study, we noted that I. scapularis OATP (IsOATP4056) that has a predicted Mock controls were treated with the same amount of solvent used for the preparation of XA.Open circles represent mock (M) and closed circles represent xanthurenic acid (XA)-treated A. phagocytophilum infected ticks.Each circle represents data from samples generated from one tick.Horizontal lines in the graphs represent the mean value of the data points.The mRNA levels of these genes were normalized to tick beta-actin mRNA levels.P value from student's t-test is shown.molecular mass of ~ 100 kDa was detected at above 250 kDa in an immunoblot performed with EL-6 and EL-2 antibody 36 .Treatment with deglycosylation mix did not impact the molecular mass of IsOATP4056 suggesting that this protein could exist in an oligomerized form or in other posttranslational-modified form 36 .Haemaphysalis longicornis OATP (GenBank Acc.No. KAH9381876) is an ortholog of IsOATP4056.The epitope region that was selected for the generation of EL-6 antibody is highly conserved in H. longicornis OATP (GenBank Acc.No. KAH9381876).Therefore, the observation of H. longicornis OATP (GenBank Acc.No. KAH9381876) at more than 250 kDa in an immunoblot suggests that this OATP may also be present in oligomerized and/or posttranslational-modified form.The observation of band above 250 kDa when SDS-PAGE analysis was performed with both 8% or 12% gel and in non-reducing and no-boiling conditions further supports that H. longicornis OATP (GenBank Acc.No. KAH9381876) may exist as an oligomerized form.In addition, the observation of multiple bands in both H. longicornis and I. scapularis total protein lysates (performed in both non-reducing and reducing conditions) is consistent with the observation of mammalian OATPs that forms dimers/homo or hetero-oligomers 45,46 .Furthermore, the presence of N-myristoylation sites, casein kinase II phospho sites, N-glycosylation sites, cAMP-and cGMP-dependent phospho sites or protein kinase C phospho sites indicate www.nature.com/scientificreports/multiple kinds of posttranslational modifications alone or in combination could also contribute for the increased molecular mass of H. longicornis OATP (GenBank Acc.No. KAH9381876).
Our previous studies reported that there is a strong interplay between OATPs and tryptophan pathway [34][35][36][37][39][40][41] . We notd that exogenous addition of XA upregulated isoatp4056 expression 40 .Knockdown of kynurenine amino transferase (KAT, enzyme that catalyzes formation of XA), via RNAi impacted isoatp4056 expression 40 .We also reported that XA indirectly could facilitate transcriptional activation of isoatp4056 gene at the promoter level 40 .These data prompted us to explore whether XA has any impact on H. longicornis OATP's expression. Ou results indicated differential regulation of H. longicornis OATPs.Interestingly, the expression of H. longicornis OATP (GenBank Acc.No. KAH9381876.1) was upregulated upon exogenous treatment with XA.These results indicate a conserved role for XA and OATP pathway in tick-A. phgocytophilum interactions. Soe H. longicornis OATPs (GenBank Acc.Nos.KAH9381028.1 and KAH9381025.1) were noted to be downregulated in the presence of exogenous XA.However, H. longicornis OATPs (GenBank Acc.Nos.KAH9381028.1 and KAH9381025.1) transcript levels were not altered upon A. phagocytophilum infection when compared to the levels noted in the uninfected controls.The significance of the downregulation of these genes in the presence of exogenous XA is not known.Future studies on these aspects would provide more details on the role of XA in different physiological processes in these ticks.
We noted significantly decreased A. phagocytophilum loads in I. scapularis ticks fed on mice passively immunized with EL-6 antibodies 36 .In addition, we noted significant decrease in A. phagocytophilum loads in ISE6 tick cells upon treatment with EL-6 antibody 36 .The observation of reduced A. phagocytophilum burden in H. longicornis ticks upon treatment with EL-6 antibody further supports our previous observations with I. scapularis ticks and ISE6 tick cell line 36 .Moreover, these data support that IsOATP4056 and KAH9381876.1 that are orthologs in I. scapularis and H. longicornis ticks, respectively, aids in the survival/colonization of A. phagocytophilum in both these ticks.Our future studies are aimed in deciphering significance of OATP-tryptophan pathways in H. longicornis-pathogen interactions.
Haemaphysalis longicornis ticks are dramatically expanding their colonies in the United States.Studies like these are not only important to address some of the important vector biological questions but also could lead us in the development of strategies to target H. longicornis ticks and pathogens they transmit.In summary, our study suggests that treatment with EL-6 antibody could be an ideal strategy to target A. phagocytophilum colonization in different ticks.

Bacteria
Anaplasma phagocytophilum isolate NCH-1 (obtained from BEI Resources, NIAID, NIH), referred as A. phagocytophilum, was used throughout this study.Anaplasma phagocytophilum was maintained in HL-60 cells and isolated from these cells as described 37,42 .Briefly, infected HL-60 cells were centrifuged for 10 min at 2300 × g at 4 °C and the pellet was washed twice with sterile 1× phosphate buffer saline.Cells were then re-suspended in 4 ml 1× PBS and subjected to 8 quick pulses of sonication (8 s burst interspersed with 8 s rest periods using an ultrasonic sonicator on an amplitude setting of 30).Cells were then passed through 27-gauge needles for 6-8 times followed by freezing at − 80 °C for 15 min.After thawing, cells were centrifuged for 5 min at 700 × g at 4 °C and the supernatant was transferred to a fresh tube.The supernatant was centrifuged for 5 min at 1000 × g at 4 °C and the resulting supernatant was transferred to another fresh tube.This supernatant was then centrifuged for 10 min at 2300 × g at 4 °C.The pellet of host-cell free A. phagocytophilum (dense core, DC) was resuspended in 100 μl of 1× PBS and used for in vitro infection.

Ticks
Haemaphysalis longicornis ticks (larvae, nymphs, adults) used in this study were obtained from BEI resources, CDC.Tick rearing was conducted in an Environmental Chamber from Parameter Generation and Control, USA.The incubator was set at 23 ± 2 °C with 94% relative humidity and 14:10 light:dark conditions.Ixodes scapularis (larvae) were obtained from BEI resources, CDC, and fed on uninfected C57BL/6J mice.Fed larvae were allowed to molt into nymphs.Total lysates from unfed nymphs were used in the immunoblotting assays.

Antibody generation
IsOATP4056-EL-6 polyclonal antibodies used in this study were generated as described 36 at a commercial facility (GenScript, USA).The I. scapularis IsOATP4056 EL-6 epitope region was used for the generation of anti-EL6 antibody 36 .

Total RNA, DNA isolation and qRT-PCR and qPCR data analysis
Total RNA from ticks (unfed/fed) was extracted using Aurum total RNA mini kit (BioRad, USA) following manufacturer instructions 37,[47][48][49] .The cDNA was generated from total RNA using iScript cDNA synthesis kit (BioRad, USA) 37,[47][48][49] , and used as template for the amplification of H. longicornis oatps and actin.DNA from ticks was extracted using DNeasy blood and tissue kit (Qiagen, USA).Oligonucleotides for actin were used from published study 50 .All other oligonucleotides used in this study are mentioned in Supplementary Table 2. qPCR and qRT-PCR was performed using iQ-SYBR Green supermix (BioRad, USA) or 2× Universal SYBR Green fast qPCR mix (ABclonal, USA) and CFX96 touch system (BioRad, USA) 37,[47][48][49] .As an internal control and to normalize the amount of template, tick actin amplicons were quantified.The standard curves were prepared using tenfold serial dilutions starting from 1 to 0.000001 ng/μl of known quantities of respective gene fragments.

Generation of A. phagocytophilum-infected H. longicornis nymphs
Unfed uninfected H. longicornis nymphs were bathed in 100 μl of A. phagocytophilum-DC preparation for 40 min at 34 °C.Ticks were washed three times with 1× PBS and stored in the tick environmental chamber for 1 week.After day 3, 5, 7 and 10 post infection, the nymphs were washed three times with 1× PBS, and the last wash of 1× PBS was saved as a negative control sample.Individual nymphs were processed for DNA extractions.DNA samples from the individual nymphs and the last 1× PBS wash sample were analyzed by qPCR to measure the bacterial loads.

Tick experiments with xanthurenic acid (XA)
Anaplasma phagocytophilum-infected H. longicornis nymphs (day 7 post-infected) were used in this experiment.Stock of XA (10 mM) (Sigma, USA) was made in 0.5 N NaOH solution 34,37 .A 1:10 dilution of the stock was prepared in 1X PBS to a final concentration of 1 mM and used in all experiments.A mock solution was prepared in a similar way but without XA.Anaplasma phagocytophilum-infected H. longicornis nymphs were bathed in 500 μl of 100 μM XA or mock solution for 40 min at 34 °C.Ticks were washed 3 times with 1× PBS and stored in the tick environmental chamber for 24 h.Individual nymph was then processed further for DNA or RNA extractions followed by qPCR or qRT-PCR analysis to measure bacterial burden or H. longicornis oatp transcripts, respectively.

Tick experiments with EL-6 antibody
Anaplasma phagocytophilum-infected H. longicornis nymphs were bathed in 500 μl of 1× PBS containing 5 μg of EL-6 antibody or control IgG antibody for 40 min at 34 °C.Ticks were washed 3 times with 1× PBS and stored in the tick environmental chamber for 24 h.Individual nymph was then processed further for DNA extractions followed by qRT-PCR analysis to measure the bacterial loads.

Western blotting assay
Immunoblotting was performed as described 48 .Five A. phagocytophilum-infected unfed H. longicornis and five A. phagocytophilum-infected unfed I. scapularis nymphs were crushed and homogenized using pellet pestle cordless motor (Biospec, OK) and pellet pestle (VWR, USA) in modified-RIPA lysis buffer supplemented with EDTA-free protease inhibitor cocktail to generate tick lysates.Protein concentrations were determined by Bradford (BCA) protein assay kit (Pierce, USA) and as per the manufacturer's recommendations.Tick lysates (10 μg) were mixed with Laemmli sample buffer, boiled for 5 min, and resolved on 8% or 12% SDS-PAGE gel (in reducing and boiling or non-reducing and no boiling conditions).Gel was run at 110 V and stained with Ponceau-S dye, destained with water, and then transferred to nitrocellulose membrane.Ponceau-S-stained image served as loading control.Membrane was blocked with 5% BSA in 1× TBST (1X TBS, 0.05% Tween 20).EL-6 antibody was used at a dilution of 1:500 in 5% BSA in 1× TBST.HRP-conjugated goat anti-rabbit secondary antibody was used at a dilution of 1:5000 in 5% BSA in 1× TBST.Development of the chemiluminescent substrate was visualized using a BioRad ChemiDoc Touch Imaging System (BioRad, USA).

Sequence alignment and bioinformatic analysis
GenBank accession numbers for the sequences used in this study are mentioned in Supplementary Table 3.Amino acid sequence alignments for H. longicornis OATPs and orthologs from various organisms were performed using DNASTAR CLUSTALW alignment software.Sequence analyses were performed for H. longicornis OATPs with various organisms.The phylogenetic tree was constructed using the neighbor-joining method (BIONJ) using BIONJ algorithm in DNSTAR.The protein sequences for H. longicornis OATPs were downloaded from GenBank and individually analyzed at the PROSITE (http:// prosi te.expasy.org/) for the prediction of N-glycosylation, myristoylation, protein kinase C phosphorylation, casein kinase II phosphorylation and cAMP or c-GMP-dependent protein kinase phosphorylation sites and as described 37,51,52 .The number of posttranslational modification sites in H. longicornis and I. scapularis are shown in Supplementary Table 4.

Statistical analysis
Statistical significance in the data sets was analyzed using GraphPad Prism6 software (https:// www.graph pad.com/) and Microsoft Excel 2010 (https:// www.micro soft.com).A student's t-test was used to compare statistical significance between the groups, or ANOVA was used to compare the variations in the groups.P values of < 0.05 were considered significant in all analyses.

Figure 1 .
Figure 1.PCR amplification and sequence analysis of H. longicornis oatp genes.(A) PCR amplification products of H. longicornis oatps is shown.M indicates the DNA marker and NTC indicates no template control.T indicates cDNA template generated from unfed H. longicornis nymphal tick RNA.GenBank accession numbers are labeled at the top of the gel image.(B) Bar graphs (with different color shades) represents percent identity of H. longicornis OATPs amino acid sequence with ortholog proteins from Dermacentor andersoni (Da), Hyalomma asiaticum (Ha), Homo sapiens (Hs), I. scapularis (Is), Mus musculus (Mm) and Rhipicephalus sanguineus (Rs).GenBank accession numbers for all OATPs are shown in Supplementary Table3.

Figure 2 .
Figure 2. Alignment of H. longicornis OATPs with the OATP-signature sequence and analysis of posttranslational modifications on H. longicornis OATPs.(A) CLUSTALW alignment of consensus OATPsignature sequence (WxGxWWxG) with all H. longicornis OATPs.Alignment was performed with DNASTAR MEGALIGN software.Residues that match are shaded in black.(B) Number of predicted post-translational sites (shown on X-axis) in H. longicornis OATPs were analyzed at PROSITE.Histograms represent the number of Nmyristoylation, casein kinase II phosphorylation, N-glycosylation, cAMP or c-GMP-dependent protein kinase phosphorylation and protein kinase C phosphorylation sites for each OATP.Full-length H. longicornis OATP sequences were considered to determine posttranslational modification sites.

Figure 3 .
Figure 3. Expression of H. longicornis oatp transcripts at different tick developmental stages.Quantitative RT-PCR analysis showing expression of kah9381028.1 (A), kah9381027.1 (B), kah9381876.1 (C), kah9365504.1 (D), kah9380884.1 (E), kah9381025.1 (F) at different tick developmental stages in uninfected unfed H. longicornis.Each data point in nymphs and adult samples represents transcript levels noted in an individual tick.Closed circles, squares and triangles represent larvae, nymphs and adults, respectively.For larval samples, each data point represents transcript levels noted in five pooled larvae.Horizontal lines in the graphs represent the mean value of the data points.The mRNA levels of these genes were normalized to tick beta-actin mRNA levels.Statistical significance was calculated using ANOVA analysis.

Figure 4 .
Figure 4. Anaplasma phagocytophilum up-regulates expression of kah9381876.1 mRNA levels in unfed H. longicornis nymphs.(A) Schematic representation showing in vitro infection of H. longicornis nymphal ticks with A. phagocytophilum.(B) qPCR with tick DNA showing levels of A. phagocytophilum in infected H. longicornis ticks at different days post-infection.Statistical significance was calculated using ANOVA analysis.qRT-PCRanalysis showing expression of kah9381028.1 (C), kah9381027.1 (D), kah9381876.1 (E), kah9365504.1 (F), kah9380884.1 (G), kah9381025.1 (H) in unfed uninfected or A. phagocytophilum-infected H. longicornis ticks.Open circles represent uninfected (UI) and closed circles represent infected (I) ticks.Each circle represents data from samples generated from one tick.Horizontal lines in the graphs represent the mean value of the data points.The mRNA levels of these genes were normalized to tick beta-actin mRNA levels.P value from student's t-test is shown.

Figure 5 .
Figure 5. Exogenous treatment with XA differentially regulates some of the oatps in A. phagocytophiluminfected unfed H. longicornis nymphs.(A) Schematic representation showing the procedure for exogenous XA treatment (100 μM) of A. phagocytophilum infected H. longicornis nymphs.(B) qPCR with tick DNA showing bacterial burden in mock or XA-treated A. phagocytophilum-infected H. longicornis ticks.qRT-PCR analysis showing expression of kah9381028.1 (C), kah9381027.1 (D), kah9381876.1 (E), kah9365504.1 (F), kah9380884.1 (G), kah9381025.1 (H) in unfed A. phagocytophilum-infected H. longicornis ticks upon treatment with XA.Mock controls were treated with the same amount of solvent used for the preparation of XA.Open circles represent mock (M) and closed circles represent xanthurenic acid (XA)-treated A. phagocytophilum infected ticks.Each circle represents data from samples generated from one tick.Horizontal lines in the graphs represent the mean value of the data points.The mRNA levels of these genes were normalized to tick beta-actin mRNA levels.P value from student's t-test is shown.

Figure 6 .
Figure 6.EL-6 antibody-treatment reduces bacterial burden in A. phagocytophilum-infected unfed H. longicornis nymphs.(A) Schematic representation of I. scapularis OATP organization on the tick cell plasma membrane is shown.Extracellular loops are indicated with numbers from 1 to 6.The N-and C-terminal ends are labeled.Antibody generated against epitope from I. scapularis EL-6 region was used in this study.Schematics are not drawn to the scale.(B) Immunoblotting analysis with EL-6 antibody showing levels of KAH9381876.1 or IsOATP4056 (indicated by black arrow) in uninfected unfed H. longicornis and I. scapularis tick lysates, respectively is shown.M indicates protein marker.Non red: indicates non-reducing conditions.Red: indicates reducing conditions, Hl indicates H. longicornis, and Is indicates I. scapularis.Ponceau S-stained gel image for total protein profile serves as loading control in the immunoblotting analysis.(C) qRT-PCR analysis showing bacterial burden (analyzed for A. phagocytophilum p44 gene DNA levels) in control IgG or EL-6 antibodytreated A. phagocytophilum-infected unfed H. longicornis ticks.Control IgG and EL-6 antibodies were used at a concentration of 5 µg/ml.Open circles represent samples generated from control IgG-treated and closed circles represent EL-6 -treated A. phagocytophilum-infected unfed H. longicornis ticks.Each circle represents data from samples generated from one tick.Horizontal lines in the graphs represent the mean value of the data points.A. phagocytophilum p44 levels were normalized to tick actin levels.P value from student's t-test is shown.